RESUMO
Cancer stem-like cells are hypothesized to be the major tumor-initiating cell population of human cutaneous squamous cell carcinoma (cSCC), but the landscape of molecular alterations underpinning their signaling and cellular phenotypes as drug targets remains undefined. In this study, we developed an experimental pipeline to isolate a highly enriched CD133+CD31-CD45-CD61-CD24- (CD133+) cell population from primary cSCC specimens by flow cytometry. The CD133+ cells show enhanced stem-like phenotypes, which were verified by spheroid and colony formation in vitro and tumor generation in vivo Gene expression profiling of CD133+/- cells was compared and validated, and differentially expressed gene signatures and top pathways were identified. CD133+ cells expressed a repertoire of stemness and cancer-related genes, including NOTCH and NOTCH1-mediated NF-κB pathway signaling. Other cancer-related genes from WNT, growth factor receptors, PI3K/mTOR, STAT pathways, and chromatin modifiers were also identified. Pharmacologic and genetic targeting of NOTCH1, IKKα, RELA, and RELB modulated NF-κB transactivation, the CD133+ population, and cellular and stemness phenotypes. Immunofluorescent staining confirmed colocalization of CD133+ and IKKα expression in SCC tumor specimens. Our functional, genetic, and pharmacologic studies uncovered a novel linkage between NOTCH1, IKKα, and NF-κB pathway activation in maintaining the CD133+ stem SCC phenotypes. Studies investigating markers of activation and modulators of NOTCH, IKK/NF-κB, and other pathways regulating these cancer stem gene signatures could further accelerate the development of effective therapeutic strategies to treat cSCC recurrence and metastasis. Mol Cancer Ther; 17(9); 2034-48. ©2018 AACR.
Assuntos
Carcinoma de Células Escamosas/genética , Quinase I-kappa B/genética , NF-kappa B/genética , Células-Tronco Neoplásicas/metabolismo , Receptor Notch1/genética , Neoplasias Cutâneas/genética , Antígeno AC133/genética , Antígeno AC133/metabolismo , Animais , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Diaminas/farmacologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Quinase I-kappa B/metabolismo , Masculino , Camundongos Nus , NF-kappa B/metabolismo , Receptor Notch1/antagonistas & inibidores , Receptor Notch1/metabolismo , Transdução de Sinais/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Tiazóis/farmacologia , Transplante HeterólogoRESUMO
Cancer stem cells (CSC) and genes have been linked to cancer development and therapeutic resistance, but the signaling mechanisms regulating CSC genes and phenotype are incompletely understood. CK2 has emerged as a key signal serine/threonine kinase that modulates diverse signal cascades regulating cell fate and growth. We previously showed that CK2 is often aberrantly expressed and activated in head and neck squamous cell carcinomas (HNSCC), concomitantly with mutant (mt) tumor suppressor TP53, and inactivation of its family member, TAp73. Unexpectedly, we observed that classical stem cell genes Nanog, Sox2, and Oct4, are overexpressed in HNSCC with inactivated TAp73 and mtTP53. However, the potential relationship between CK2, TAp73 inactivation, and CSC phenotype is unknown. We reveal that inhibition of CK2 by pharmacologic inhibitors or siRNA inhibits the expression of CSC genes and side population (SP), while enhancing TAp73 mRNA and protein expression. Conversely, CK2 inhibitor attenuation of CSC protein expression and the SP by was abrogated by TAp73 siRNA. Bioinformatic analysis uncovered a single predicted CK2 threonine phosphorylation site (T27) within the N-terminal transactivation domain of TAp73. Nuclear CK2 and TAp73 interaction, confirmed by co-immunoprecipitation, was attenuated by CK2 inhibitor, or a T27A point-mutation of this predicted CK2 threonine phospho-acceptor site of TAp73. Further, T27A mutation attenuated phosphorylation, while enhancing TAp73 function in repressing CSC gene expression and SP cells. A new CK2 inhibitor, CX-4945, inhibited CSC related SP cells, clonogenic survival, and spheroid formation. Our study unveils a novel regulatory mechanism whereby aberrant CK2 signaling inhibits TAp73 to promote the expression of CSC genes and phenotype.